Triglyceride Assay

Colorimetric assay for the quantitative determination of Triglyceride

in plasma and serum.

CM10010303

96

For illustrative purposes only.

To perform the assay the instructions for use provided with the kit have to be used.

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Instructions for Use

Triglyceride Assay KitCatalog No. 10010303

3GENERAL INFORMATION

TABLE OF CONTENTS GENERAL INFORMATION 3 Materials Supplied

4 Precautions

4 If You Have Problems

4 Storage and Stability

4 Materials Needed but Not Supplied

INTRODUCTION 5 Background

6 About This Assay

PRE-ASSAY PREPARATION 7 Reagent Preparation

8 Sample Preparation

ASSAY PROTOCOL 9 Plate Set Up

11 Standard Preparation

12 Performing the Assay

ANALYSIS 13 Calculations

14 Performance Characteristics

RESOURCES 16 Troubleshooting

17 References

18 Related Products

18 Warranty and Limitation of Remedy

19 Plate Template

20 Notes

GENERAL INFORMATION

Materials Supplied

Catalog Number Item Quantity

10010509 Triglyceride Standard 1 vial

10010508 Triglyceride Standard Diluent 1 bottle

10010510 Triglyceride Assay Buffer 1 bottle

10010511 Triglyceride Enzyme Mixture 1 vial

400012 96-Well Plate 1 plate

400014 Plate Cover 1 cover

If any of the items listed above are damaged or missing, please contact our Customer Service department at (800) 364-9897 or (734) 975-3999. We cannot accept any returns without prior authorization.

! WARNING: Not for human or animal disease diagnosis or therapeutic drug use.

4 GENERAL INFORMATION 5INTRODUCTION

PrecautionsPlease read these instructions carefully before beginning this assay. For research use only. Not for human or diagnostic use.

If You Have ProblemsTechnical Service Contact Information

Phone: 888-526-5351 (USA and Canada only) or 734-975-3888Fax: 734-971-3641E-Mail: techserv@caymanchem.comHours: M-F 8:00 AM to 5:30 PM EST

In order for our staff to assist you quickly and efficiently, please be ready to supply the lot number of the kit (found on the outside of the box).

Storage and StabilityThis kit will perform as specified if stored as directed at -20°C and used before the expiration date indicated on the outside of the box.

Materials Needed But Not Supplied1. A plate reader capable of measuring absorbance between 530-550 nm2. Adjustable pipettes and a repeat pipettor3. A source of pure water; glass distilled water or HPLC-grade water is acceptable4. Test tubes5. 15 ml centrifuge tube6. Aluminum foil

INTRODUCTION

BackgroundTriglycerides are water-insoluble lipids consisting of three fatty acids esterified to a glycerol backbone. Triglycerides are transported in the blood as core constituents of all lipoproteins, but are major components of triglyceride-rich chylomicrons and very low-density lipoproteins (VLDL).1 A major source of triglycerides is dietary fat. Dietary fats are hydrolyzed in the gut into free fatty acids and mono- and diglycerides and then transported through the intestinal villi. After absorption through the gut, they are resynthesized into new triglycerides and assembled into chylomicrons. Triglycerides are rapidly hydrolyzed in the capillary beds by lipoprotein lipase, releasing glycerol and free fatty acids, which are absorbed by adipose tissue for storage. When required, lipases hydrolyze triglycerides from adipose tissue into fatty acids and glycerol, which enter the blood stream. Fatty acids are oxidized in the mitochondria and peroxisomes to produce energy. Triglycerides play an important role in metabolism by containing more than twice as much energy as carbohydrates and proteins. The measurement of triglyceride levels, in conjunction with other lipid assays, are useful in the diagnosis of primary and secondary hyperlipoproteinemia, dyslipidemia, and triglyceridemia. Triglyceride concentrations are also useful in the diagnosis and treatment of diabetes mellitus, nephrosis, liver obstruction, and other diseases involving lipid metabolism or various endocrine disorders.2,3,4 The most common method to determine triglyceride concentrations is by enzymatic hydrolysis of triglycerides to glycerol and free fatty acids followed by either colorimetric or fluorometric measurement of the glycerol released.5-8

6 INTRODUCTION 7PRE-ASSAY PREPARATION

About This AssayCayman’s Triglyceride Assay provides a simple, reproducible, and sensitive tool for assaying triglycerides in plasma and serum. The Triglyceride Assay uses the enzymatic hydrolysis of the triglycerides by lipase to glycerol and free fatty acids. The glycerol released is subsequently measured by a coupled enzymatic reaction system (Figure 1). The glycerol formed in reaction 1 is phosphorylated to glycerol-3-phosphate in a reaction catalyzed by glycerol kinase (eq 2). The glycerol-3-phosphate is oxidized by glycerol phosphate oxidase producing dihydroxyacetone phosphate and hydrogen peroxide (eq 3). Peroxidase catalyzes the redox-coupled reaction of H2O2 with 4-aminoantipyrine (4-AAP) and N-Ethyl-N-(3-sulfopropyl)-m-anisidine (ESPA), producing a brilliant purple color (eq 4). The absorbance is measured at 540 nm.

Triglycerides

Quinoneimine dye + 4H2O (4)Peroxidase

Glycerol + Fatty Acids (1)

Glycerol PhosphateOxidase

Glycerol Kinase

LipoproteinLipase

Glycerol + ATP

Glycerol-3-Phosphate + O2

2H2O2 + 4-AAP + ESPA

Glycerol-3-Phosphate + ADP (2)

Dihydroxyacetone Phosphate + H2O2 (3)

Figure 1. Triglyceride Assay Scheme

PRE-ASSAY PREPARATION

Reagent Preparation1. Triglyceride Standard - (Catalog No. 10010509)

Each vial contains a 1,000 mg/dl solution of Triglyceride Standard. It is ready to use as provided to prepare the standard curve. Sufficient Triglyceride Standard is provided to prepare three standard curves.

2. Triglyceride Standard Diluent - (Catalog No. 10010508)The vial contains 10 ml of a salt solution. This solution should be thawed and stored at room temperature. This Standard Diluent solution is used to prepare the triglyceride standards and may be stored for six months at room temperature until it is ready for use.

3. Triglyceride Assay Buffer - (Catalog No. 10010510)The vial contains 15 ml of 50 mM sodium phosphate buffer, pH 7.2. This solution should be thawed and stored at room temperature. This buffer is used to prepare the triglyceride enzyme solution. The assay buffer may be stored for at least six months at room temperature until it is ready for use.

4. Triglyceride Enzyme Mixture - (Catalog No. 10010511)The vial contains a lyophilized enzyme mixture. Reconstitute the contents of the vial with 1 ml of UltraPure water. Transfer the reconstituted solution to a 15 ml centrifuge tube wrapped in aluminum foil. Add 14 ml of the assay buffer to the reconstituted solution and mix by inversion. NOTE: A portion of the 14 ml should be used to rinse any residual solution from the vial. This solution is now ready to use in the assay. If the entire solution is not used at one time, the solution should be stored at 4°C. Do NOT Freeze! The solution is stable for one month when stored at 4°C; a slight pink discoloration may occur but will have no affect on the assay performance.

8 PRE-ASSAY PREPARATION 9ASSAY PROTOCOL

ASSAY PROTOCOL

Plate Set UpThere is no specific pattern for using the wells on the plate. A typical layout of triglyceride standards and samples to be measured in duplicate is given below in Figure 2. We suggest you record the contents of each well on the template sheet provided (see page 19).

1-8 = StandardsS = Samples

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Figure 2. Sample Plate Format

Sample PreparationPlasmaTypically normal human plasma has triglyceride concentrations in the range of 40-160 mg/dl (male) or 35-135 mg/dl (female).9

1. Collect blood using an anticoagulant such as heparin, EDTA, or citrate. 2. Centrifuge the blood at 700-1,000 x g for 10 minutes at 4°C. Pipette off the top

yellow plasma layer without disturbing the white buffy layer. Store plasma on ice. If not assaying the same day, freeze at -80°C. The plasma sample will be stable for one month while stored at -80°C.

3. Plasma does not need to be diluted before assaying.

SerumTypically normal human serum has triglyceride concentrations in the range of 40-160 mg/dl (male) or 35-135 mg/dl (female).9

1. Collect blood without using an anticoagulant.2. Allow blood to clot for 30 minutes at 25°C.3. Centrifuge the blood at 2,000 x g for 15 minutes at 4°C. Pipette off the top yellow

serum layer without disturbing the white buffy layer. Store serum on ice. If not assaying the same day, freeze at -80°C. The serum sample will be stable for one month while stored at -80°C.

4. Serum does not need to be diluted before assaying.

10 ASSAY PROTOCOL 11ASSAY PROTOCOL

Pipetting Hints• It is recommended that an adjustable pipette be used to deliver reagents to the

wells.• Before pipetting each reagent, equilibrate the pipette tip in that reagent

(i.e., slowly fill the tip and gently expel the contents, repeat several times).• Do not expose the pipette tip to the reagent(s) already in the well.

General Information• All reagents except samples must be equilibrated to room temperature before

beginning the assay.• The final volume of the assay is 160 µl in all wells.• The incubation temperature is at room temperature.• It is not necessary to use all the wells on the plate at one time.• It is recommended that the standards and samples be assayed at least in duplicate.• Monitor the absorbance at 530-550 nm using a plate reader.

Standard PreparationTake eight clean test tubes and label them 1-8. Add 200 µl of the Triglyceride Standard Diluent (Catalog No. 10010508) to tubes 2-8. Add 400 µl of Triglyceride Standard Diluent (Catalog No. 10010508) to tube 1. Add 100 µl of Triglyceride Standard (Catalog No. 10010509) to tube 1 and mix thoroughly. The concentration of Tube 1 is 200 mg/dl (2.26 mmol/L), from which serial dilutions will be made. Serially dilute the triglycerides by removing 200 µl from tube 1 and adding it to tube 2; mix thoroughly. Next, remove 200 µl from tube 2 and place it into tube 3; mix thoroughly. Repeat this process for tubes 4-7. Tube 8 only has Triglyceride Standard Diluent and is used as the blank. We recommend that you store these diluted standards for no more than one to two hours. See Table 1 below for the triglyceride concentrations of the serial dilutions.

Tube Triglyceride Concentration (mg/dl)

1 200

2 100

3 50

4 25

5 12.5

6 6.25

7 3.125

8 0

Table 1. Preparation of Triglyceride Standards

12 ASSAY PROTOCOL 13ANALYSIS

Performing the Assay1. Triglyceride Standard Wells - Add 10 µl of standard (tubes 1-8) per well in the

designated wells on the plate (see suggested plate configuration, Figure 2, page 9). 2. Sample Wells - Add 10 µl of sample (either undiluted plasma or serum) to two or

three wells. NOTE: The amount of sample added to the well should always be 10 µl.3. Initiate the reaction by adding 150 µl of diluted enzyme buffer solution to each well.4. Carefully shake the microtiter plate for a few seconds to mix. Cover with the plate

cover.5. Incubate the plate for 15 minutes at room temperature.6. Read the absorbance at 530-550 nm using a plate reader.

ANALYSIS

Calculations1. Calculate the average absorbance of each standard and sample.2. Subtract the absorbance value of standard 8 (0 mg/dl) from itself and all other values

(both standards and samples). This is the corrected absorbance.3. Graph the corrected absorbance values (from step 2 above) of each standard as a

function of the final triglyceride concentration (mg/dl) (see Table 1, page 11). A typical triglyceride standard curve is shown in Figure 3 on page 15.

4. Calculate the values of triglyceride samples using the equation obtained from the linear regression of the standard curve by substituting the corrected absorbance values for each sample into the equation.

Triglycerides (mg/dl) = (Corrected absorbance) - (y-intercept)

Slope ][

14 ANALYSIS 15ANALYSIS

Performance CharacteristicsPrecision:When a series of sixteen human serum samples were assayed on the same day, the intra-assay coefficient of variation was 1.34%. When a series of sixteen human serum samples were assayed on six different days under the same experimental conditions, the inter-assay coefficient of variation was 3.17%.

Assay Range:Under the standardized conditions of the assay described in this booklet, the dynamic range of the kit is 0-200 mg/dl triglyceride.

Representative Triglyceride Standard CurveThe standard curve, presented on page 15, is an example of the data typically provided with this kit; however, your results will not be identical to these. You must run a new standard curve - do not use this data to determine the values of your samples.

0.100

0.200

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0.000

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sorb

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m)

y = 0.003x + 0.0027

r2 = 0.9999

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100 140

0.400

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0.600

Figure 3. Triglyceride standard curve

16 RESOURCES 17RESOURCES

RESOURCES

Troubleshooting

Problem Possible Causes Recommended Solutions

Erratic values; dispersion of duplicates/triplicates

A. Poor pipetting/techniqueB. Bubble in the well(s)

A. Carefully tap the side of the plate with your finger to remove bubbles

B. Be careful not to splash the contents of the wells

No triglyceride was detected in the sample

Triglyceride concentration was too low or the sample was too dilute

Do not dilute samples and re-assay

Sample absorbance values are above highest point in standard curve

Triglyceride concentration was too high in the sample or the sample was too concentrated

Dilute samples with assay buffer and re-assay. NOTE: Remember to account for the dilution factor when calculating the triglyceride concentration.

References1. Cole, T. G.; Klotzsch, S. G.; McNamara, J. R. Measurement of Triglyceride

Concentration in Handbook of Lipoprotein Testing. N. Rifai, et. al., Ed. AACC Press. Washington DC, 115, (1997).

2. Fredrickson, D.S., Levy, R.I., and Lees, R.S. Fat transport in lipoproteins - an integrated approach to mechanisms and disorders. New England Journal of Medicine 276(1), 34-42 (1967).

3. Rifai, N., Bachorik, P.S., Albers, J.J. Lipids, Lipoproteins, and Apolipoproteins in Tietz Fundamentals of Clinical Chemistry. C.A. Burtis, E.R. Ashwood, Ed. WB Saunders Company, Philadelphia, PA, 462-493 (2001).

4. Wahlefeld, A.W. in Methods of Enzymatic Analysis, Vol. 5. H.U. Bergmeyer, Ed. Academic Press, New York, NY, 1831-1835, (1974).

5. Bucolo, G. and David, H. Quantitative determination of serum triglycerides by the use of enzymes. Clin. Chem. 19(5), 476-482 (1973).

6. Fossati, P. and Prencipe, L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28(10), 2077-2080 (1982).

7. McGowan, M.W., Artiss, J.D., Strandbergh, D.R., et al. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin. Chem. 29(3), 538-542 (1983).

8. Mendez, A.J., Cabeza, C., and Hsia, S.L. A fluorometric method for the determination of triglycerides in nanomolar quantities. Anal. Biochem. 156, 386-389 (1986).

9. Deska-Pagana, K. and Pagana, T.J. in Mosby’s Diagnostic and Laboratory Test Reference. Seventh Edition, Mosby, St. Louis, 937-938 (2005).

18 RESOURCES 19RESOURCES

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Warranty and Limitation of RemedyCayman Chemical Company makes no warranty or guarantee of any kind, whether written or oral, expressed or implied, including without limitation, any warranty of fitness for a particular purpose, suitability and merchantability, which extends beyond the description of the chemicals hereof. Cayman warrants only to the original customer that the material will meet our specifications at the time of delivery. Cayman will carry out its delivery obligations with due care and skill. Thus, in no event will Cayman have any obligation or liability, whether in tort (including negligence) or in contract, for any direct, indirect, incidental or consequential damages, even if Cayman is informed about their possible existence. This limitation of liability does not apply in the case of intentional acts or negligence of Cayman, its directors or its employees.Buyer’s exclusive remedy and Cayman’s sole liability hereunder shall be limited to a refund of the purchase price, or at Cayman’s option, the replacement, at no cost to Buyer, of all material that does not meet our specifications.Said refund or replacement is conditioned on Buyer giving written notice to Cayman within thirty (30) days after arrival of the material at its destination. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims hereunder with respect to said material.For further details, please refer to our Warranty and Limitation of Remedy located on our website and in our catalog.

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NOTES

This document is copyrighted. All rights are reserved. This document may not, in whole or part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form without prior consent, in writing, from Cayman Chemical Company.©07/30/2008, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed in U.S.A.

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